Abordagem Independente De Cultivo

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Abordagem Independente De Cultivo DANIEL SAITO CARACTERIZAÇÃO DAS COMUNIDADES BACTERIANAS ASSOCIADAS ÀS INFECÇÕES ENDODÔNTICAS: ABORDAGEM INDEPENDENTE DE CULTIVO Tese apresentada à Faculdade de Odontologia de Piracicaba, da Universidade Estadual de Campinas, para obtenção do Título de Doutor em Biologia Buco-Dental, Área de Concentração Microbiologia e Imunologia. Orientador: Prof. Dr. Reginaldo Bruno Gonçalves PIRACICABA 2007 i FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA DA FACULDADE DE ODONTOLOGIA DE PIRACICABA Bibliotecário: Marilene Girello – CRB-8a. / 6159 Saito, Daniel. Sa28c Caracterização das comunidades bacterianas associadas às infecções endodônticas: abordagem independente de cultivo. / Daniel Saito. -- Piracicaba, SP : [s.n.], 2007. Orientador: Reginaldo Bruno Gonçalves. Tese (Doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. 1. Reação em Cadeia da Polimerase. 2. RNA Ribossômico 16S. 3. Polimorfismo de Fragmento de Restrição. I. Gonçalves, Reginaldo Bruno. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título. (mg/fop) Título em Inglês: Characterization of bacterial communities associated with endodontic infections: culture-independent approach Palavras-chave em Inglês (Keywords): 1. Polymerase Chain Reaction. 2. 16S Ribosomal RNA. 3. Restriction Fragment Length Polymorphism. Área de Concentração: Microbiologia e Imunologia Titulação: Doutor em Biologia Buco-Dental Banca Examinadora: Marli de Fátima Fiore, Luis Eduardo Aranha Camargo, José Francisco Höfling, Sérgio Roberto Peres Line, Reginaldo Bruno Gonçalves Data da Defesa: 14-12-2007 Programa de Pós-Graduação em Biologia Buco-Dental ii iii Dedico este trabalho À minha querida esposa Cris, por todo o seu amor, a compreensão e a dedicação À nossa filhinha recém-nascida Marieva, que trouxe nova inspiração às nossas vidas Aos meus pais Wilson e Tsai, pelo amor incondicional de uma vida inteira À minha irmã Lin, por todo o carinho e a amizade compartilhados desde a nossa infância Aos meus avós, que sempre me acompanham na mente e no coração iv AGRADECIMENTOS Ao Prof. Dr. Reginaldo Bruno Gonçalves, grande amigo e orientador, cuja experiência e sabedoria guiaram meus passos no doutorado, contribuindo imensamente para a minha formação acadêmica. Ao Prof. Dr. José Francisco Höfling, chefe do Laboratório de Microbiologia e Imunologia, amigo de longa data e que sempre me apoiou nos momentos mais difíceis dessa caminhada. Aos amigos do Curso de Pós-Gradução em Biologia Buco-Dental Alessandra Castro Alves, Ana Paula Amoras, Bruna de Araújo Lima, Cristiane Duque, Fernando Zamuner, Flávia Sammartino Mariano, Gustavo Obando Pereda, Iza Alves Peixoto, Janaína de Cássia Orlandi Sardi, Marlise Inêz Klein, Paula Cristina Aníbal, Rafael Nóbrega Stipp, Regianne Umeko Kamiya, Rita de Cássia Mardegan, Ruchelle Nogueira, Sérgio Eduardo Braga Cruz, Thaís de Cássia Negrini, Vivian Furletti e aos técnicos do Laboratório de Microbiologia e Imunologia Anderson Laerte Teixeira e Wilma C. Ferraz, pela grande amizade e pelos momentos de alegria e descontração durante toda a nossa convivência. À Prof. Dra. Siu Mui Tsai, chefe do Laboratório de Biologia Molecular e Celular do CENA – USP, por ter cedido espaço precioso em seu laboratório durante o período de dois anos para as análises de seqüenciamento de DNA. À minha querida esposa Cristiane Pereira Borges Saito, por ter me auxiliado na coleta de amostras clínicas. À doutoranda Fabiana de Souza Cannavan e ao Dr. José Elias Gomes e ao Dr. Jorge Luiz Mazza Rodrigues, pelo suporte dado às técnicas de PCR, clonagem e seqüenciamento de 16S rDNA. Ao Prof. Renato de Toledo Leonardo, por ter concedido espaço na Clínica de Endodontia da Faculdade de Odontologia de Araraquara – UNESP para a coleta de amostras clínicas e, principalmente, pela amizade e confiança sempre depositadas em minha pessoa. v Ao Prof. Dr. Alexandre Augusto Zaia, por ter gentilmente concedido espaço no Atendimento de Urgência da Faculdade de Odontologia de Piracicaba – UNICAMP para a coleta de amostras clínicas. Ao Prof. Sérgio Roberto Peres Line, pelos inúmeros ensinamentos passados a mim durante o meu doutorado e, acima de tudo, pela grande amizade construída ao longo desses anos. Ao Prof. Dr. Luiz Lehmann Coutinho e a todos os colegas do Laboratório de Biotecnologia Animal da ESALQ – USP, por estarem sempre dispostos a dividir a infraestrutura e o conhecimento sobre a técnica de PCR em Tempo Real. Ao Prof. Dr. Terence Lee Marsh e a todos os colegas do Center for Microbial Ecology, Michigan State University, pelos ensinamentos sobre a técnica de T-RFLP e ecologia microbiana, e por terem cordialmente recebido a mim e a minha esposa. À Prof. Dra. Vânia Maria Maciel Melo do Centro de Ciências da Universidade Federal do Ceará e à Prof. Dra. Vivian Pellizari do Instituto de Ciências Biológicas - USP, colegas de trabalho da Michigan State University, pelo apoio e companheirismo dados a mim e a Cris durante o nosso intercâmbio. Ao Dr. Éderson da Conceição Jesus, colega de trabalho da Michigan State University, por ter me repassado informações importantíssimas sobre a técnica de T-RFLP. Aos integrantes da minha banca de qualificação, Prof. Dr. José Francisco Höfling, Prof. Dr. Ricardo Della Coletta e Dr. Rogério Castilho Jacinto, pelas importantes considerações feitas ao meu trabalho de doutorado. A Faculdade de Odontologia de Piracicaba e a todos os integrantes do corpo administrativo, sem o qual esta tese de doutorado não poderia ser realizada. A CAPES e a FAPESP, pelo apoio financeiro durante todo o meu Curso de Pós-Graduação e por possibilitarem o meu intercâmbio no exterior. Agradeço sinceramente a todos. vi RESUMO A presente tese teve como objetivo a caracterização das comunidades bacterianas associadas às infecções endodônticas pelo emprego de técnicas moleculares independentes de cultivo. Ao todo, foram analisadas amostras intra-radiculares provenientes de 34 elementos dentários associados a infecções endodônticas. A análise de bibliotecas clonais de DNA ribossomal 16S (16S rDNA) permitiu a identificação de 2 a 14 filotipos bacterianos (espécies) por elemento dentário (média= 9,6), perfazendo um total de 46 filotipos distintos. Dentre estes, 9% foram considerados previamente desconhecidos e classificados taxonomicamente como novos membros da ordem Clostridiales. Espécies reconhecidamente endodônticas dos gêneros Bacteroides, Campylobacter, Eubacterium, Peptostreptococcus, Selenomonas, Treponema e Veillonella foram detectadas, assim como representantes de gêneros menos freqüentemente descritos, como Burkholderia, Filifactor e Megasphaera. O emprego da técnica quantitativa de PCR em Tempo Real, possibilitou a detecção de P. gingivalis, T. forsythia e a coexistência de ambas em 24%, 56% e 18% dos pacientes avaliados, respectivamente. Nenhuma correlação significativa foi evidenciada entre os níveis de ambas as espécies, individualmente ou em conjunto, e a presença de sintomatologia dolorosa. O uso de T-RFLP na avaliação da estrutura das comunidades bacterianas revelou um total de 123 (endonuclease HhaI) e 122 (endonuclease MspI) fragmentos de restrição terminais (T-RFs) distintos, com médias de 20,8 e 20,0 T-RFs por elemento dentário, respectivamente. Aproximadamente 50% dos fragmentos detectados apresentaram-se, no máximo, em 2 pacientes, indicando uma alta variabilidade na composição microbiana. As análises de clusterização e de estatística multivariada não revelaram diferenças significativas nas comunidades bacterianas entre os grupos de estudo assintomático, sensível ao toque e sintomático. De modo geral, os resultados obtidos reiteraram o conceito de que a microbiota associada às infecções endodônticas é essencialmente polimicrobiana, altamente variável entre indivíduos, e constituída predominantemente por bactérias anaeróbias Gram-positivas do filo Firmicutes. As espécies P. gingivalis e T. forsythia, embora relativamente prevalentes nas infecções endodônticas, não apresentaram correlação significativa com o desenvolvimento de sintomatologia dolorosa. Por fim, a ausência de agrupamentos de perfis bacterianos quanto aos parâmetros sintomatológicos sugere que a estrutura das comunidades bacterianas intra-radiculares não possui influência significativa no desenvolvimento da dor de origem endodôntica. Palavras-chave: infecção endodôntica, 16S rDNA, biblioteca clonal, PCR em Tempo Real, T-RFLP. vii ABSTRACT The objective of the present study was to characterize the bacterial communities associated with endodontic infections by use of culture-independent molecular techniques. Overall, 34 intraradicular samples from teeth harboring endodontic infections were evaluated. 16S ribosomal DNA (16S rDNA) clone library analysis allowed the identification of 2 to 14 bacterial phylotypes (species) per tooth (mean= 9.6), with a total of 46 distinct phylotypes. Among the latter, 4 (9%) were considered previously unreported and further taxonomically classified as members of the order Clostridiales. Well-known endodontic representatives of Campylobacter, Eubacterium, Peptostreptococcus, Selenomonas, Treponema e Veillonella were detected, as well as members of less frequently reported genera, such as Burkholderia, Filifactor and Megasphaera. The application of the Real Time PCR technique permitted the detection of P. gingivalis, T. forsythia and a coexistence of both in 24%, 56% e 18% of the subjects, respectively. No significant correlations were evidenced among the levels of P. gingivalis and T. forsythia, individually or conjointly, and spontaneous
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